Cytology Kristin M. Canga, RVT

References: Bassert and Thomas: Clinical Textbook for Veterinary Technicians, 8th edition

Hendrix and Sirois: Laboratory Procedures for Veterinary Technicians, 5th edition

Reading Assignments

Laboratory Procedures book: Chapter 9, pp: 287-336

CTVT: Pp: 374 – 375, Vaginal Cytology Pp: 418 – 422, Cytology through end of chapter

Labs for this chapter: Ear Cytology Vaginal Cytology Estrous Cycle cell project

Cytology - Definition

Microscopic examination of individual cells or a small group of cells that have been ________________________ from a tissue or structure.

Cells come from: _____________________________ _____________________________ _____________________________ _____________________________

Mast Cell Tumor

Histopathology

Greek – Histos – _____________________________ Pathos – _____________________________

Histopathology observes cells in relation to other cells and evaluates the architecture of those tissues.

Involves complex steps and specialized equipment

Use of _____________________________ Studies the manifestation of

_____________________________ Microscopic _____________________________changes in

tissue More invasive – surgical, biopsy

Why do we do cytology?

Differentiate:_____________________________ lesion (non-

tumor)_____________________________mass (tumor)

Rapid resultsLess expensiveNot as invasiveMay be more helpful for certain samples

Mast cell tumorNot as a replacement for

histopathology…adjunct diagnostic tool.

How do we collect samples?

1: ___________________ 2: ___________________ 3: ___________________ 4: ___________________collection

(aspirate and non-aspirate) 5: ___________________ 6: ___________________ 7: ___________________

1: Swabs

Collected when imprints, scrapings and aspirates cannot be made.

Can be taken from ___________________, nasal passages, rectum, ___________________, ___________________ or vagina.

Cells collected are those that have exfoliated from the surface.

You can identify: Yeast, bacteria, WBC, RBC, ear mites, other parasites, fungi, neoplastic cells and epithelial cells.

Swabs – Sample Collection

Materials – Sterile cotton swab, clean glass slide, 0.9% sterile saline, Diff-Quick

Technique:Pre-moisten a swab with saline (Optional in

some cases)Place pre-moistened swab into/onto the cavity

Use gentle pressure to avoid damage to cellsGently roll swab onto clean glass slide in a

single stroke in 2-3 rows down the length of the slide

Allow to air dry (wave in air)Apply heat if an ear swabStain with Diff-Quik

2: Scrapings

Taken from ___________________ lesions on animal Prepared from tissues collected during

___________________ or surgery Collect many cells from tissue Advantageous when lesion is firm or yields few

cells Disadvantages – difficult to collect and only

___________________ Used to reflect a secondary bacterial infection,

external parasites, or inflammation-induced tissue dysplasia

Technique External Parasites:

Supplies: Scalpel blade, mineral oil, slide, gloves

Pinch are of crusted or hairless skin between fingers

Scrape the surface at a 45, or 90 degree angle until capillary oozing (light bleeding)

Place on clean clean slide with mineral oil and examine under microscope

Perform on more than one area if multiple lesions

What external parasites can we find with a skin scraping?

Biopsy Scrapings

Biopsy samples: Supplies: Scalpel blade, clean slide, stain,

glovesExpose fresh edge of tissueBlot sample to its nearly dryHold blade at 90 degree angle and scrap

across the tissueSpread onto a clean slide – if too thick

make a compression slideAir dry Stain

Scraping

3: Imprints (Impression Smear)

Prepared from external lesions on the ___________________ animal or from tissues removed during ___________________or ___________________

Collects ___________________ cells than scrapings

Greater amount of ___________________ May hinder the veterinarian in making an

accurate diagnosis of ___________________ You may find: Mast cells, bacteria, Yeast,

WBC’s, RBC’s, neoplastic cells

Tzanch (Tzanck) Preparation

Imprint preparation – external lesions Procedure:

4-6 clean glass slidesSlide 1 – touch to un-prepped lesionSlide 2 – Prep with saline, gently debride and

lightly clean lesion, then touch slide to lesionSlide 3 – Fully debride lesion and re-imprint

with slideSlide 4 – Imprint the underside of scab if

presentAir dry all slidesStain

Imprint Sample

Taken from tissues collected during surgery or necropsy

Procedure:Expose a fresh edge on a small piece of

tissueBlood and tissue should be first removed

from the surface of the lesionTouch the tissue repeatedly in rows in

single file on a clean slideAir dry the slide before stainingConsider making multiple slides

4: Fine Needle Collection Collected from ___________________ Lymph nodes, nodular lesions and internal organs Advantage: Avoid superficial ___________________ Disadvantage: Fewer cells collected Methods:

___________________ ___________________

Supplies: Gloves Syringe Needle Slide Stain Surgical prep/alcohol

Site Preparation

Surgical preparationIf sample collected from:

______________________________________

Non-surgical preparation – any other sampleClean area with alcohol swab

Fine Needle Aspirate (FNA) Technique:

21-25 –gauge needle (22g commonly used) 3-20 ml syringe (12 ml is useful size) Stabilize mass Insert needle Retract plunger to create negative pressure and draw cells Redirect needle several times

Maintain negative pressure to prevent contamination in lg. masses

Do not exit the mass Remove needle from mass Remove syringe from needle Fill syringe with air Reattach needle Gently force sample from needle onto clean slide Make several slides if possible

Non-aspirate Procedure

___________________ technique Stabilize mass Insert needle (22 gauge)

May have syringe barrel attached without the plunger Redirect needle several times

Do not exit the mass https://www.youtube.com/watch?v=0eH5NRzK7QE

Remove needle from mass Remove syringe barrel (if used) from needle

Fill syringe with air Re-attach needle Expel material onto clean glass slide Repeat

5: Tissue Biopsy

Sampling of a piece of tissue for ___________________and/or ___________________ examination

Techniques: Abrasion with blade, needle aspiration, excision and endoscope-guided biopsy. Varies by ___________________ Consider: location, accessibility and nature

Preparation: Clip hair Do NOT cleanse or scrub the site or disrupt scales, crusts

or surface debris. May offer valuable diagnostic information

Tissue Biopsy

Once specimen is collected: Gently remove specimen by grasping margin of tissue

with fine forceps If collected by endoscopy flush specimen with sterile

saline from tip of endoscope Blot on paper towel to remove excess blood Place on small piece of wooden tongue depressor and

allow to dry Immerse or float specimen with tongue depressor

specimen side down in fixative

Tissue Biopsy - Histopathology

If for histopathologic examination tissues are often fixed in 10% __________________ phosphate-buffered ___________________. Tissue no > than 1 cm Fluid jar should contain ____x the specimen’s

volume Large tissues can be transferred to a smaller jar with

less formalin after fixed for 24 hours.

Wedge Biopsy

Elliptic specimen commonly obtained with a scalpel

Advantage: ___________________ specimen Excise entire lesion or the wedge is taken from an

area of the lesion Excise through ___________________ zone to normal

tissue Pathology technician can trim specimen to provide

the pathologist with a slide showing ___________________ tissue, ___________________ zone and ___________________ tissue.

Wedge Biopsy

Punch Biopsy

Advantage: speed and procedure Keyes cutaneous biopsy punches (4, 6, 8-mm) 6 / 8 mm punches require one or two sutures 2-3 specimens should be collected If multiple lesions samples should be collected

from the various lesions

Punch Biopsy Procedure Clip biopsy site being careful to not cause skin inflammation Select appropriate biopsy punch Have suture ready if necessary Gently rotate the biopsy punch in one direction until the

punch blade has sectioned the tissue Grasp the margin of tissue with a pair of fine forceps or flush

tissue with saline onto a small piece of wooden tongue depressor

Allow the tissue to dry or blot on paper towel Place the tissue into a formalin jar, specimen side down on a

tongue depressor Changes in specimen can occur within 1 minute after the biopsy is

obtained Specimens should be allow to fix for at least 24 hours before

processing. Specimens should remain at room temperature for at least 6

hours before exposing to extreme cold.

6: Centesis

Definition: Introduction of a ___________________ into any body cavity or organ for the purpose of removing ___________________

____________________________________ – collection of fluid from abdominal cavity

____________________________________ – collection of fluid from the thoracic cavity

____________________________________ – aspiration of urine from the urinary bladder

____________________________________ – aspiration of the pericardial sac to retrieve fluid

____________________________________ – fluid collection from around the spinal column, from within joints and from around the eye

Centesis

Thoracocentesis Animal in standing position Needle inserted into the 7th or 8th intercostal

space along the cranial aspect of the rib Abdominocentesis

May be performed in a standing animal or with pt. in lateral recumbency.

Needle introduced into the ventral abdomen to the right of the midline, 1-2 cm caudal to umbilicus

Centesis - Procedures

For peritoneal or pleural fluid the site should be aseptically prepared the site and equipment

A portion of collected fluid should be collected into an EDTA tube to prevent clotting.

21-gauge needle attached to a 60 ml syringe Prepare several smears from fluid as soon as it is

collected Record total volume collected, color and turbidity Determine total nucleated cell counts, cell types,

and their morphologic characteristics General anesthesia required for some

procedures such as collection of cerebrospinal fluid

Abdominocentesis

Thoracocentesis

Centesis – Color and Turbidity

Influenced by __________________ concentration and cell numbers Discoloration may be from contamination with blood, recent or old

hemorrhage, inflammation or both. Perforation of superficial vessels during collection may result in

reddish discoloration. Peripheral blood contamination or recent hemorrhage result in clear

supernatant and red (erythrocyte rich) sediment after centrifugation. Inflammation may cause a degree of turbidity reflecting leukocyte

numbers. Color = off-white, cream, red-cream or dirty brown

7: Transtracheal / Bronchial Wash

Cytologic evaluation of samples obtained from trachea, bronchi, or bronchioles May assist with diagnosis of ____________________________

disease of animals ____________________________________ Technique

Involves insertion of needle into trachea through cricothyroid membrane to infuse saline and collect fluid when the animal coughs

Laryngeal area is clipped and aseptically prepared Local anesthetic – 2% Lidocaine

____________________________________ Technique Placing an endotracheal tube in an anesthetized patient

and collecting fluid through a jugular or urinary catheter Preferred in small and/or fractious animals Animal is lightly anesthetized

TRANSTRACHEAL WASH - VIDEO

http://www.youtube.com/watch?v=QOhGoSgMB5c

Bronchoalveolar lavage / Nasal Flush

Bronchoalveolar lavage – orotracheal technique used to collect samples from the lower respiratory tract. Bronchoscopy is preferred method but requires

bronchoscope Nasal flush is used for collecting cytologic samples

from the nasal cavity Investigates diseases of the upper airway

Initially, only a small amount of saline infused will be harvested

Cells of interest may be present if the animal coughs after initial collection

Sample Preparation – Orotracheal

All fluid released during coughing should be collected Place fluid in a sterile tube with notation on site of

collection If sample contains a small amount of mucus

centrifuge at a low speed and prepare smears. If sample contains a large amount of mucus it

may not need to be centrifuged. Nucleated cell counts are not generally

performed, but cell numbers are subjectively recorded

Mucus often appears as eosinophilic to purple strands.

Epithelial cells are principal cell type

Smear Preparations – solid masses

Several methods Experience level of person preparing and

characteristics of the sample influence choice of technique

Combination of techniques is suggested: ____________________________________ Preparation ____________________________________ Technique ____________________________________ Smear

Compression “Squash” Preparation

Transfer sample to clean slide near the frosted edge and toward the middle of the slide.

Gently placing a second slide (spreader slide) over aspirate perpendicular to the first slide.

Allow the sample to spread for a few seconds. Spreader slide (slide 2) is quickly and smoothly

slid across prep slide; do not place pressure on spreader of slide.

Air dry spreader slide (slide 2) and stain Modification: Less tendency to rupture cells

Lay second slide over aspirate, rotate second slide 45 degrees, lift upward.

Compression Slide Procedure (pg 301-302)

Combination Technique

Spray sample onto the middle of a clean, glass microscope slide (prep slide)

Place the prep slide on a flat surface and pull another slide (spreader slide) backward at a 45 degree angle to the first slide until it makes contact with approximately 1/3 of the aspirate.

Spread forward as if making a blood smear The spreader slide is then placed horizontally over the

back third of the aspirate at a right angle to the prep slide. The weight of the slide usually spreads the material

If needed, slide the spreader slide across the prep slide in a quick, smooth motion.

It is important to leave the center portion unspread. This allows for viewing of a high concentration of cells.

Combination Preparation (pg 303)

Starfish Smear

Transfer sample to the center of a clean slide

Use the tip of a needle to “drag” the sample outward from the center with the point of the needle in a starfish shape

Vary the length and direction of each “drag” through the sample

Does not tend to damage cells

Air dry the slide before staining by gently waving it in the air

Preparation of smears from fluid samples

Smears should be prepared immediately following collection

Collect in ______________ tubes Smear technique is influenced by cellularity,

viscosity, and homogeneity of fluid. Line smear

Concentrates cells when fluid cannot be concentrated by centrifugation (fluids of low cellularity and translucent)

Place drop of fluid on a clean glass slide Use blood smear technique except spreading slide

is raised directly upward ¾ of the way through the smear = a line of much higher concentrated cells.

Wedge smear – like blood smear

How do we fix and stain our samples?

Advantageous to incorporate a separate cellular fixative Preferred 95% methanol Must be fresh and not contaminated with cellular

debris 2-5 minutes (greater is ok)

Romanowsky Wrights, Giesma, Diff-Quik

Papanicolaou New Methylene Blue

Romanowsky Stains

Inexpensive and readily available (Diff-quick, Wright’s) Stain organisms and the cytoplasm of cells Nucleolar detail sufficient for differentiating neoplasia

and inflammation, but Papanicolaou stains are better Each stain has a unique procedure Thinner smears, or samples with lower total protein

concentration of the fluid generally need less time needed in stain. (And vice versa)

Amount of time in stain (number of dips) will also depend on age of stain. Very variable, be consistent.

Keep stains covered and away from light

Romanowsky Stains

Fixative 95%

Methanol

Eosin – Acidic PHStains basic

components of cells

Methylene Blue-

Alkaline PHStains acidic components

Distilled Water

New Methylene Blue

Used when critical nuclear detail must be visualized

Stains cytoplasm weakly, if at all Gives nuclear and nucleolar detail Useful for when you suspect malignancy

Papanicolaou (Pap stain)

Delicate Multichromatic stain used for smear preparation Give excellent nuclear and cytoplasmic detail Commonly used to diagnose neoplasia Does not stain cytoplasm as strongly as Romanowsky Multiple steps and considerable time

5 dyes in 3 solutions Specimen must be wet fixed (before cells dry)

Spray with wet fixative or place in ethanol immediately after preparation

With ethanol must be on protein coated slide to prevent cells from falling off slide when immersed.

NOT commonly done in clinic

Papanicolaou (Pap stain)

How to prevent staining problems Always use new, clean slides Pre-cleaned slides should be wiped with alcohol

before use Fresh stains and buffer solutions (if required)

should be used Fix samples immediately after air drying unless

being sent to a outside lab Consult with the laboratory prior to taking samples

if sending to an outside lab. Do not touch surface of slide with hands

Submission of Slides to Outside Lab

When in-house evaluation of cytology does not provide sufficient information

Done by Clinical Pathologist Contact lab to find out protocol for

preparation and shipping of samples Submit 2-3 air-dried, unstained slides and

2-3 dried, stained slides Also send EDTA and/or sterile serum

tubes of samples if applicable Make sure you use slide mailers

Initial Microscopic Evaluation Perform initial evaluation of cytology with low magnification –

100x (see fig. 9-27) Determine if all areas are adequately stained

Be consistent! Large objects – clusters, parasites, crystals and fungal hyphae

will be visible if present on 10x Initial evaluation should be used to characterize the cellularity

and composition Record relative numbers of cells present High powered examination (400x – 450x) should be performed

to evaluate and compare individual cells and further characterize the types of cells present.

Oil immersion to identify and differentiate evidence of inflammation vs. neoplasia

Cytology report should contain: ____________________________________ ____________________________________ Relative Proportions

Inflammation

Normal _________________________ response to tissue damage or invasion by microorganisms.

Cytology samples from inflammatory sites are characterized by the presence of white blood cells _________________________ _________________________

Occasionally eosinophils and lymphocytes may be present.

Fluid samples are often turbid and white or pale yellow.

Characterizing inflammation

_________________________ (purulent): large numbers of neutrophils (usually > ____% of the total nucleated cell count). Neutrophils are the first WBC to the site of inflammation.

_________________________ or _________________________ : >15% macrophages present. Fungal and parasitic infection often manifest with this

presentation. _________________________ : > 10% of eosinophils +

increased neutrophils. Usually found with parasitic infection, but may also be

present in some neoplastic disorders. Once designated as inflammatory the cells must also

be evaluated for evidence of degeneration and presence of microorganisms.

What type of inflammation is seen here?

What type of inflammation is shown here?

Nuclear Changes in Inflammatory Cells

_________________________ – represents slow cell death Condensed dark nucleus that may fragment

Pyknotic Cells

Nuclear Changes in Inflammatory Cells

_________________________ – Nuclear fragmentation Chromatin is distributed irregularly throughout

the cell.

Nuclear Changes in Inflammatory Cells

_________________________ – Rapid cell death, dissolution of nuclear fragments Septic (bacterial) inflammatory reactions

Inflammatory cells that contain _________________________ microorganisms

Appears as a swollen, ragged nucleus without an intact nuclear membrane

_________________________ staining ability

Karyolysis

Yellow Arrow!!

To review:

Septic Inflammatory Cells

Inflammatory cells that contain _________________________ microorganisms are referred to as _________________.

Additional material may include erythrocytes, parasites, and fungal organisms.

Septic Inflammatory Cells (Note phagocytized bacteria present in cytoplasm)

Neoplasia Neoplastic specimens normally contain homogenous

populations of a single cell type. Indicated when the cells are of the same tissue

origin. First be differentiated as benign or malignant. Benign – _________________________ with no criteria

of _________________________ present in nucleus of cells. Uniform appearance

Malignant – cells that display at least ______ abnormal _________________________ configurations.

Exception - _________________________ is also present or only a few cells display malignant characteristics.

Nuclear Criteria of Malignancy ( Table 9-2)

_________________________ – variation in size of nuclei _________________________ – Variability in the size and

shape of the same cell type High or variable nucleus / cytoplasm ratio Increased ______________ activity – Any increase in the

presence of mitotic figures or cells that are not dividing equally. (Mitosis is rare in normal tissue.)

Coarse ____________ Pattern – May appear ropy or cordlike

Nuclear molding - deformation of nuclei by other nuclei within the same cell or adjacent cells

_________________________ - Multiple nuclei within a cell Nucleoli that vary in size (_________________________ ),

shape (angular nucleoli), and number (multiple nucleoli)

Staging and Grading of Neoplasia

How widespread is the disease Tumor – invasiveness,

size Nodes – none,

regional, distant involvement

Metastasis – none or evidence Usually areas with

dense capillary beds Lymphnodes, liver, lungs

How mature How well differentiated

are the cells

How immature Undifferentiated =

danger

Staging Grading

Nuclear Criteria of Malignancy

Anisokaryosis – unusual variation in overall size of nuclei (Page 313 definition is a mistake…)

Pleomorphism

Variability in the size and shape of same cell type.

Primary Types of Tumors

Epithelial cell tumors – carcinoma or adenocarcinoma Highly cellular and often exfoliate in clumps

Primary types of tumors: Mesenchymal

Sarcoma - usually less cellular, exfoliate singly or in wispy spindles

Pleomorphism Multinucleation Coarse chromatin

pattern Macronuclei

Primary Types of Tumors: Round cell

Exfoliate very well but are usually not in clumps or clustersHistiocytomaLymphomaMast cell tumorsPlasma cell tumorsMelanoma

Round Cell Tumor ComparisonLymph nodes:

Normal Abnormal

Peritoneal and Pleural Fluid

Normal Colorless to transparent and slightly turbid. Odorless

Gross discoloration and increased turbidity may be the result of increased cell numbers and/or protein concentration.

Total nucleated cell count (TNCC) – same methods as for CBC. Normal is less than 10,000 nucleated cells/μl Nucleated cells are categorized as neutrophils,

large mononuclear cells, lymphocytes, eosinophils, and any other nucleated cells

Few erythrocytes – estimate on smear TNCC and total protein values are classified into

transudate, modified transudate or exudate.

Transudates vs. Exudates

Modified Transudate Relatively low to moderate total cell counts,

predominately low to moderate total cell counts Results from leakage of lymphatic fluid Low numbers of inflammatory cells

Exudates Fluids with increased

cellularity and protein concentrations because of inflammation, infection or trauma

Absolute numbers of neutrophils > 85%

Macrophages, neutrophils, bacteria, degenerative or toxic neutrophils

Transudate An effusion characterized

by low protein concentration and low total nucleated cell counts.

Secondary to congestive heart failure, liver failure, kidney failure

Thin fluid Only a few neutrophils or

macrophages

Cytology of Lymph Nodes

May show evidence of: Inflammation – lymphadenitis Hyperplasia – benign neoplasia Mixed – inflammatory and neoplastic present Neoplasia – lymph node cells with abnormal

nuclear features Metastasis – neoplastic cells from other body

tissues that spread to the lymph nodes Usually collected by FNA – always take from two

lymph nodes Compression technique and stain with Romanowsky

Reactive Lymph Node

Normal – Predominant cell type is small, mature lymphocyte

Lymph nodes responding to antigenic stimulation also contain small, mature lymphocytes

Plasma cells, lymphoblasts, and intermediate lymphocytes are more abundant than in a normal lymph node.

Occasional Mott cells (plasma cells containing secretory vesicles of immunoglobulin) Note: these can appear similar to vacuoles

Reactive Lymph Node

Mott Cell

Malignant Neoplasia - lymphoma

Predominately characterized by lymphoblasts Mitotic figures are common Macrophages present Plasma cells scarce May see other neoplastic cells – mast cells,

carcinoma cells, sarcoma cells, histiocytes Lymph node samples may also contain

metastatic cells from other body parts

Lymphoma

Ear Cytology Normal:

Cornified squamous epithelial cells Negiliable evidence of hemorrhage or

inflammation Common abnormal findings:

_________________________ (Malassezia) _________________________ _________________________

Ear Cytology Procedure

Collect sample AU prior to medicating or cleaning ears.

Use cotton swab to recover debris from external ear canal

Gently roll onto clean glass slide (Thin layer = best)

If examining for mites, do not let slide dry, examine unstained, immediately.

If examining for bacteria, fungi, or malassezia, allow to air dry or heat fix with lighter.

Use DiffQuick stain, and examine on Oil Immersion Lens.

Vaginal Cytology

Used in conjunction with history and clinical examination in determining the stage of estrous cycle in bitches and queens.

Variation in terminology used to describe cell types

Stages are brought about by changes in _________________________ and _________________________ concentrations

Repeat examinations may be necessary In addition to neutrophils and erythrocytes, a

variety of squamous epithelial cells are also seen in vaginal cytology preps

Vaginal Cytology Procedure

The objective is to obtain samples from the vagina, not the vulva.

At a relatively steep angle, gently insert cotton swab several inches past the vulva (even in small dogs)

After 1-2 inches of swab have been inserted, adjust to approximately a 45° angle and continue gentle insertion.

After insertion, rotate the swab 2-3 full rotations to allow cells to adhere to swab.

Remove swab and gently roll on clean glass slide. (usually done in two parallel tracks on top and bottom portion of slide.

Stain with DiffQuick immediately

Vaginal Cytology Procedure

Insertion of the swab

Vaginal Cytology Procedure – Application to slide

Parabasal Cells

__________________ epithelial cells seen on a typical vaginal smear

________________ or nearly round and have a high nuclear to cytoplasmic ratio.

Prevelant during ______________ and _______________

Absent during ______________

Intermediate Cells

Diameter 2-3x that of a parabasal cell May contain ______________ nuclei Prevalent during all stages of the cycle except

______________ Vary in size Some are a rounded shape and others have a

polygonal shape

Superficial Cells ______________ cells seen on a vaginal smear Polygonal in shape in distinctly flat, may appear

folded Often seen in large ______________ or strings Nuclei are ______________ or pyknotic (small and dark) Without nuclei are referred to as being “fully

______________ ” Not normally seen during ______________ Large numbers of only superficial cells are a defining

characteristic of cytologic estrus.

Other Cells Neutrophils Red Blood Cells Bacteria Usually of no pathologic significance

Anestrus

No vulvar swelling Does not attract male dogs Predominately non-cornified squamous epithelial

cells Primarily ______________ and Intermediate cells No ______________ Lasts < 4.5 months

Proestrus

Vulvar swelling Slightly bloody discharge Males attracted Female not receptive Lasts 4-13 days, beginning of ______________ Cells are non-cornified with nucleus to large

superficial squamous epithelial cells Decreased ______________ Increased ______________ Early – High RBC’s Late – cornified epithelial cells and pyknotic nuclei

Proestrus

Estrus

Lasts about 4-13 days Vulvar swelling Discharge pinkish to straw colored Becomes whiter as ______________ approaches Female accepts male All ______________ squamous epithelial cells, usually

______________ ______________ absent Small numbers of ______________ present

Estrus

Metestrus aka diestrus

2-3 months, coming out of heat (estrus) Little/no vulvar swelling Female not receptive to males Decreased ______________ cells Increased non-cornified cells ______________ increase until about day three, then

decrease Often difficult to differentiate from ______________ .

Metestrus aka diestrus

Vaginitis and Metritis

Inflammation of vagina or uterus results in pinkish-white

Usually without vulvar swelling or clinical signs of proestrus or estrus

Vaginal swab revels non-cornified squamous epithelial cells and a large number of neutrophils w/free or ____________________________ bacteria.

Semen Evaluation

Upon collection, avoid exposing semen samples to marked changes in ______________ (especially cold), water, disinfectants or variations in pH.

Laboratory equipment and supplies used in semen collection and examination should be clean, dry and warmed to approximately 98.6°C (37°F)

Stains and diluents should be warmed as well.

Characteristics

Volume Gross appearance Wave motion Microscopic motility Spermatozoal concentration Ratio of live/dead spermatozoal

concentration Assessment of morphologic features Presence of foreign cells or material

Volume of Ejaculate

Measured with a ____________________________ flask ______________ variation occurs Method of collection greatly influences volume

obtained, gross appearance, and spermatozoal concentration

Three portions: sperm- __________watery secretion, sperm-___________ fraction, and sperm-_________ fraction.

Bucks, bulls, rams and toms: all three fractions are collected together

Boars, dogs and stallions the ______ fraction may be collected separately

Gross Appearance of Ejaculate

Record _____________ and color of the sample

Catagories include: Thick, creamy, opaque, milky

opaque, opalescent milky, and watery white

Sperm Motility

Assessed ____________________________ Must be handled carefully for meaningful results

Do not expose to non-isotonic fluids or destructive chemicals

1. Wave motion Classified as very good, good, fair and poor based on the swirling

activity in a drop of semen on a microscope at low magnification. Decreases as sperm concentration decreases

2. Motility (Correlated with ______________ ) Relatively dilute drop of semen under cover slip, examined at

100X magnification Dilute with warm physiologic saline or fresh buffered 2.9 Sodium

Citrate solutions are dependable. Add cover slip Percentage of motile sperm are classified as very good, good,

fair, poor, corresponding to rapid linear activity. A sample should have about 60% moderately active spermatozoa

Semen Evaluation

Semen Samples diluted and counted with a hemactyometer.

Sample settles for a few minutes and checked for homogenous distribution of spermatoza

Sperm concentration varies with method of collection

Staining with a vital dye permits discrimination between live and dead spermatozoa

Eosin/Nigrosin mixture stain

Small drop of warm stain is gently mixed with a small drop of semen on a warm microscope slide

After a few seconds of contact make a smear like you would a blood smear.

Sperm Concentration Live/Dead Sperm Ratio

Sperm Morphology

Observe 100 to 500 cells and not percentage of abnormal sperm Abnormalities divided into head, mid-piece and tail. Problems are categorized as primary or secondary.

Other cells in semen: normal semen contains few (if any) WBC’s, RBC’s, or epithelial cells and no bacteria and fungi

Semen Collection

Semen collected from dogs for breeding soundness exams, artificial ____________________________ , or to examine prostatic fluid for culture or cytology in cases of prostatic ______________

Semen collected for insemination can be used fresh, cooled and shipped to another location, or frozen for long term storage

“Teaser” bitch in estrus – ideal Especially if male is nervous or if first time collection Male is not allowed to mount Latex collection cone with attached tube commonly

used.

Semen Collection

Vaginal swab from estrous bitch Good to keep several swabs frozen until needed

Digital pressure and massage most common Electro ejaculation and AV (artificial vagina)

collection are also common collection techniques.

We’re Done with Cytology!